Inclined Sedimentation for Selective Retention of Viable Hybridomas in a Continuous Suspension Bioreactor

Abstract

The continuous separation of nonviable hybridoma cells from viable hybridoma cells by using a narrow rectangular channel that is inclined from the vertical has been investigated experimentally. The effectiveness of the settler in selectively retaining viable hybridomas in the bioreactor while permitting the removal of nonviable hybridomas has been shown to depend on the flow rate through the settler. Intermediate flow rates through the settler have been found to provide the highest removal of nonviable hybridomas relative to viable hybridoma retention. At high dilution rates through the chemostat, over 95% of the viable cells could be partitioned to the bottom of the settler while over 50% of the nonviable cells are removed through the top of the settler. This successful separation is due to the significantly larger size of the viable hybridomas than the nonviable ones. A continuous perfusion experiment was performed in which an external inclined settler was used to retain virtually all of the viable hybridomas in the culture, while selectively removing from the culture approximately 20% of the nonviable cells that entered the settler. A stable viable cell concentration of 1.0 x 10(7) cells/mL was achieved, as was an antibody productivity of over 50 micrograms/(mL.day). These represent 3- and 6-fold increases, respectively, over the values obtained from a chemostat culture without cell retention.

Separation of CHO cells using hydrocyclones

“…In both cases (decrease in diameter or in cell density), the terminal settling velocity of the cells decreases, favoring the separation of dead cells in the overflow stream. A similar behaviour has been observed in inclined settlers and Centritech centrifuges (Batt et al 1990;Takagi et al 2000; Lipscomb , which are equipment that also separate particles based on their terminal velocity. The cells were also monitored within the first 48 h after passage through the hydrocyclone by fluorescence microscopy to evaluate any possible induction of apoptosis.…”

“…These data give evidence that the hydrocyclones showed a preferential retention of viable cells, separating non-viable cells preferentially in the overflow stream, which in a perfusion run would be the harvest stream. Acoustic filters also presented preferential separation of viable hybridoma cells, and this was credited to the decrease of the mean diameter of dead cells (Batt et al 1990;Lipscomb et al 2004). However, it is probable in the present case that not only the alterations in size, but also the changes in density have influenced cell separation.…”

Apoptosis-resistant E1B-19K-expressing NS/0 myeloma cells exhibit increased viability and chimeric antibody productivity under perfusion culture conditions

“…This has been observed for cells cultivated in batch (Ramirez and Mutharasan, 1990), fed-batch (de Tremblay et al, 1993;Robinson et al, 1994), chemostat (Boraston et al, 1984), and perfusion modes (Al-Rubeai et al, 1992;Banik and Heath, 1995;Batt et al, 1990;de la Broise et al, 1991;Johnson et al, 1996;Hansen et al, 1993;Park and Ryu, 1994;Tokashiki and Takamatsu, 1993;Trampler et al, 1994), as well as in cells treated with cell-cycle blocking agents (Al-Rubeai and Emery, 1990;Jayme, 1991;Mercille and Massie, 1998;Oh et al, 1995;Øyaas et al, 1994a,b;Reddy and Miller, 1994;Suzuki and Ollis, 1990;Takahachi et al, 1994). Indeed, specific antibody production has been shown to be cell cycle dependent and optimum in the G 1 and/or G 2 /M phase (Cazzador and Mariani, 1993;Kromenaker and Srienc, 1991;Linardos et al, 1992;Richieri et al, 1991;Suzuki and Ollis, 1989).…”

Mammalian cell retention devices for stirred perfusion bioreactors

“…In practice the maximum measured velocity for total retention of viable cells was only approximately 50% of the predicted value (Batt et al, 1990) proba- bly because the prediction assumes a plug flow profile rather than the true parabolic velocity profile within each channel. Additional deviation from the ideal capacity can be attributed to disruptive convective flows due to non-uniform heating or cooling.…”

“…Selective retention of viable mammalian cells was demonstrated by Batt et al (1990) as a function of flow-rate, with the difference between viable and nonviable cell retention being up to 50% within a range of intermediate flow rates.…”